Among the various pluripotent stem cells, induced pluripotent stem cells (iPSC)-generated by ectopic expression of four embryonic reprogramming genes in somatic cells-provide a source of patient-specific stem cells without the ethical limitations associated with embryonic stem cells (ESC). However, we and others have reported that iPSC are incompletely reprogrammed, and we found that the degree of reprogramming decreases with the age of the donor tissue. Our overall research goal is to improve the reprogramming of iPSC in order to produce high-quality patient-specific histocompatible tissues for transplantation. Because older patients are more likely to benefit from the clinical applications of iPSC, there is a significant need to comprehensively evaluate iPSC derived from older donor cells (A-iPSC) and identify additional pluripotent reprogramming factors that can reverse the negative impact of aging on A-iPSC reprogramming. Multiple reports have proposed that p53 is a negative regulator of somatic cell reprogramming, and that elimination of p53 increases iPSC reprogramming efficiency. However, basal p53 levels are elevated in ESC and iPSC compared with somatic cells, leading us to propose an innovative hypothesis-that iPSC reprogramming is incomplete due to insufficient inhibition of elevated p53 by p53 inhibitory factors. We further hypothesized that A-iPSC may be unable to activate these putative p53 inhibitory factors to overcome the elevated p53 during iPSC reprogramming. As a result, p53 cannot be completely restrained, and the residual p53 activity stimulates a constitutive DNA damage response that mimics what normally occurs only in response to a genotoxic insult. A chronic DNA damage response (via p53-dependent and -independent pathways) has also been observed during normal aging of somatic cells, and can lead to the complete loss of the DNA damage response during cancer development. We posited that epigenetic destruction of the entire DNA damage response would improve A-iPSC reprogramming and quality. In preliminary studies, we examined the function of two putative p53 inhibitory factors and used these factors to identify pluripotent regulatory factors that can correct the aging-related epigenetic landscape and improve the quality of A-iPSC. In this proposal, we will examine the function of these pluripotent regulatory factors by conducting a comprehensive comparative analysis of these iPSC types. In Aim 1, we will determine the mechanism by which the pluripotent regulatory factors reverse the aberrant DNA damage response and negative effects of aging on the A-iPSC phenotype. In Aim 2, we will determine the functional effects of the pluripotent regulatory factors by comparing the pluripotency and differentiation potential of each cell line in several in vitro and in vivo tests. Ultimately, our findings will provide critical insight into how we can improve the quality of patient-specific stem cells derived from aged patients for use in tissue regeneration and transplantation.